Previous studies had strongly indicated that phosphorylation of the heavy chains of Acanthamoeba myosin II inactivated its actin-activated ATPase activity by altering the conformation of the myosin II filaments. By developing new assay procedures, it has been possible to keep myosin II monomeric and thus demonstrate that, in contrast to filamentous myosin II, phosphorylated monomeric myosin II retains full enzymatic activity. This result supports the conclusion that regulation of myosin II by phosphorylation occurs at the level of the filament. Immunofluorescence and immunoelectron microscopy have shown the myosin I heavy chain kinase is strongly localized to the plasma membrane. Kinase binds with high affinity to plasma membranes. The C-terminal half of the kinase contains about half of the total phosphorylation sites, these are still autophosphorylatable, and autophosphorylation enhances the myosin I kinase activity of the fragment. However, in contrast to the native enzyme, autophosphorylation of the fragment is not enhanced by acidic phospholipids.